The present invention relates to a synergistic composition useful for qualitative screening of phosphate solubilizing microorganisms. More particularly, the present invention provides synergistic composition for qualitative screening of phosphate solubilizing microorganisms, based upon visual observation.
A large portion of inorganic phosphates applied to soil as fertiliser is rapidly immobilised after application and becomes unavailable to plants (Yadav K S, Dadarwal K R (1997) Phosphate solubilisation and mobilisation through soil microorganisms. In: Dadarwal K R (ed): Biotechnological approaches in soil microorganisms for sustainable crop production. Jodhpur, India: Scientific Publishers, pp. 293-308. Thus, the release of insoluble and fixed forms of phosphorus is an important aspect of increasing soil phosphorus availability. Seed or soil inoculation with phosphate solubilizing bacteria is known to improve solubilisation of fixed soil phosphorus and applied phosphates resulting in higher crop yields. [Abd-Alla M H (1994) Phosphatases and the utilisation of organic phosphorus by Rhizobium leguminosarum biovar viceae. Lett Appl Microbiol 18:294-296; Jones D L, Darrah P R (1994) Role of root derived organic acids in the mobilisation of nutrients from the rhizosphere. Plant Soil 166:247-257; Leyval C, Barthelin J (1989) interactions between Laccaria laccata, Agrobacterium radiobacter and beech roots: Influence on P, K, Mg and Fe mobilisation from mineral and plant growth. Plant Soil 17:103-110; Yadav K S, Dadarwal K R (1997) Phosphate solubilisation and mobilisation through soil microorganisms. In: Dadarwal K R (ed). Biotechnological approaches in soil microorganisms for sustainable crop production. Jodhpur, India: Scientific Publishers, pp. 293-308j. Several authors attribute the solubilisation of inorganic insoluble phosphate by microorganisms to the production of organic acids and chelating oxo acids from sugars [Leyval C, Barthelin J (1989) Interactions between Laccaria laccata, Agrobacterium radiobacter and beech roots: Influence on P, K, Mg and Fe mobilisation from mineral and plant growth. Plant Soil 17:103-110; Yadav K S, Dadarwal K R (1997) Phosphate solubilisation and mobilisation through soil microorganisms. In: Dadarwal K R (ed): Biotechnological approaches in soil microorganisms for sustainable crop production. Jodhpur, India: Scientific Publishers, pp. 293-308]. Therefore, most of the quantitative tests to assay the relative efficiency of the phosphate solubilizing bacteria are based on the lowering of pH, due to production of organic acids into the surrounding medium [Bajpai P D, Sundara Rao W V B (1971) Phosphate solubilizing bacteria II. Extracellular production of organic acids by selected bacteria solubilizing insoluble phosphates. Soil Sci Plant Nutr 17:44-45; Gaind S, Gaur A C (1989) Effect of pH on phosphate solubilisation by microbes. Curr Sci 58:1208-1211; Johnston H W (1952) The solubilisation of phosphate: the action of various organic compounds on dicalcium and tricalcium phosphate. NZJ Sci Technol 33:436-444; Rose R E (1957) Techniques of determining the effect of microorganisms on insoluble inorganic phosphates. N.Z.J Sci Technol 38:773-780; Sethi R P, Subba Rao N S (1968) Solubilisation of tricalcium phosphate and calcium phytase by soil fungi. J Gen Appl Microbiol 14:329-331]. The initial isolation of phosphate solubilisers is usually made by using a medium suspended with insoluble-phosphates such as tri-calcium phosphates [Tilak K V B R (1993) Bacterial Fertilisers. New Delhi, India: Indian Council of Agricultural Research]. The production of clearing zones around the colonies of the organism is an indication of the presence of phosphate solubilizing organisms. Such cultures are isolated and extent of phosphate solubilisation determined quantitatively, using biochemical methods (Tilak K V B R (1993) Bacterial Fertilisers. New Delhi, India: Indian Council of Agricultural Research; Subba Rao N S (1993) Biofertilizers in agriculture and forestry. Oxford and IBH Publishing Company Pvt. Ltd., New Delhi: Oxford and IBH Publishing Company Pvt Ltd.].
Phosphate solibilising microorganisms are routinely screened by a plate assay method using Pikovskaya (PVK containing (gm/Lt): glucose, 10; Ca3 (PO4)2, 5; (NH4)2SO4, 0.5; NaCl, 0.2; MgSO4.7H2O, 0.1; KCl, 0.2; yeast extract, 0.5; MnSO4.H2O, 0.002 and FeSO4.7H2O, 0.002) agar [Pikovskaya R I (1948) Mobilisation of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17:362-370]. Other phosphate solibilising media known in the prior art besides PVK are as follows containing (gm/Lt): 1. Glucose, 10; CaHPO45, yeast extract, 0.5; KCl, 0.2; MgSO4.7H2O, 0.1; MnSO4, trace; and FeSO4.7H2O, trace [Sundara Rao, W V M, Sinha M K (1963) Phosphate dissolving organisms in soil and rhizosphere. Ind. J. Agri. Sci. 33, 272-278].
2. Glucose, 10; Ca3 (PO4)2, 5; (NH4)2SO4, 1; MgSO4.7H2O, 0.5; KCl, 0.2; yeast extract, 0.2; MnSO4.H2O, trace and FeCl3, trace [Halder A K, Mishra A K, Chakarbartty P K (1991) Solubilisation of inorganic phosphates by Bradyrhizobium Ind. J. Exp. Biol. 29, 28-31].
3. Sucrose, 5; CaHPO4, 5; MgSO4.7H2O 0.5; KNO3, 1; KCl, 0.5; and FeSO4.7H2O, 0.011 [Wenzel, C. L., Ashford, A. E. and Summerell, B. A. (1994) Phosphate solubilizing bacteria associated with protoid roots of seedlings of waratoh (Telopea speciosissama.) New Phytol. 128, 487-496].
4. Mannitol, 10: Hydroxyapetite, 2; NH4Cl2, 1; MgSO4.7H2O, 0.5; KCl, 0.2; biotin, 0.001; calcium pantothenate. 0.001; MnSO4.H2O, 0.002 and FeCl3, 0.002 [Abd-Alla M H (1994) Phosphatases and the utilisation of organic phosphorus by Rhizobium leguminosarum biovar viceae. Lett Appl Microbiol 18:294-296].
The test of the relative efficiency of isolated strains is carried out by selecting the microorganisms which are capable of producing a halo/clear zone on plate due to the production of organic acids into the surrounding medium [Pikovskaya R I (1948) Mobilisation of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17:362-370].
However, as the reliability of this halo-based technique is questioned as many isolates which did not produce any visible halo/zone on agar plates could solubilise various types of insoluble inorganic phosphates in liquid medium a modified PVK medium using bromophenol blue (BPB), to improve the clarity and visibility of the yellow-coloured halo has not necessarily improved the plate assay [Gupta R, Singal R, Shankar A, Kuhad R C Saxena R K (1994) A modified plate assay for screening phosphate solubilizing microorganisms. J Gen Appl Microbiol 40:255-260]. Thus, the existing plate assay fails where the halo is inconspicuous or absent. Contrary to indirect measurement of phosphate solubilisation by plate assay, the direct measurement of phosphate solubilisation in broth assay always resulted into reliable results [Nautiyal C S (1999) An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 170:265-270]. It was suggested that microbes from soil may be screened in National Botanical Research Institute""s phosphate growth medium (NBRIP) broth assay for the identification of most efficient phosphate solubilisers [Johri J K. Surange S Nautiyal C S (1999) Occurrence of salt, pH and temperature-tolerant, phosphate-solubilizing bacteria in alkaline soils. Curr Microbiol 39:89-93; Nautiyal C S, Bhadauria S, Kumar P, Lal H, Mondal R, Verma D (2000) Stress induced phosphate solubilisation in bacteria isolated from alkaline soils. FEMS Microbiol Lett 182:291-296]. NBRIP contained (per liter): glucose, 10 g; Ca3 (PO4)2, 5 g; MgCl2.6H2O, 5 g; MgSO4.7H2O, 0.25 g; KCl, 0.2 g, and (NH4)2SO4, 0.1 g [Nautiyal C S (1999) An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 170:265-270].
However, screening large number of isolates for phosphate solubilisation by quantitative methods requires investment of time, labour and chemicals. Thus BPB a blue coloured dye which decolorises due to drop in pH of the medium was used, as an indicator to quickly evaluate the level of phosphate solubilisation based upon visual observations.
Therefore, it is desirable to formulate a defined media for screening phosphate solubilizing microorganisms, based upon visual observations, to quickly evaluate the level of phosphate solubilisation.
The main object of the present invention is to provide a synergistic composition which is based upon visual observations, and not on quantitative analysis involving investment of time, labour and chemicals, to quickly evaluate the level of phosphate solubilisation
Accordingly the present invention provides a synergistic composition containing BPB designated, xe2x80x9cNBRI-BPBxe2x80x9d which comprises:
In an embodiment of the present invention, the ingredients used in the synergistic composition of the present invention may be of commercial grade. However, the use of purer ingredients such as that of laboratory grade will result in better reproducible results.
The synergistic composition of the present invention is not a mere admixture of the ingredients used resulting in aggregation of their properties but a synergistic mixture having enhanced properties resulting in identifying phosphate solubilizing microorganisms.
In one embodiment, the invention provides a qualitative method for easy and fast screening of phosphate solubilizing comprising a synergistic growth medium composition containing essential bromophenol blue (BPB) in the range of 0.01 to 0.1 gm/Lt.
In another embodiment of the present invention a kit for the screening of phosphate solubilizing microorganisms comprising BPB in a growth medium, comprising:
The synergistic composition of the present invention may be prepared by any one of the known methods.